Direct application of non-toxic crosslinking reagents to resist progressive spinal degeneration and deformity

ABSTRACT

A method of treatment of native, non-denatured tissue to increase resistance to tearing, fissuring, rupturing, and/or delamination, comprising the step of: contacting at least a portion of the tissue with an effective amount of a reagent that increases crosslinks in the tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.11/346,464, filed on Feb. 2, 2006, which is a continuation-in-part ofapplication Ser. No. 10/786,861, filed on Feb. 24, 2004, which claimsthe benefit of U.S. Provisional Application Ser. No. 60/498,790, filedon Aug. 28, 2002, and which is a continuation-in-part of applicationSer. No. 10/230,671, filed on Aug. 29, 2002, which claims the benefit ofU.S. Provisional Application Ser. No. 60/316,287, filed on Aug. 31,2001.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for treatment of tissue, forexample, collagenous tissue, where a deleterious mechanical loadingenvironment contributes to the degradation of the tissue. In variousembodiments, the present invention relates to a method for treatment ofload supporting fibrous tissues, such as degenerated intervertebraldiscs and surrounding tissues, degenerative knee meniscus, prevention ofextrusion or expulsion of materials or devices implanted such as nucleusreplacement or augmentation, to methods and devices to improve theresistance to tearing of the tissue and to prevent disc herniation, andto methods to improve fatigue resistance, to resist ongoing deformingforces and curve progression in scoliosis as well as other progressivespinal deformities, to non-toxic crosslinking reagents that areeffective fatigue and tear inhibitors and joint stabilizers, and tomethods and devices for improving the environment for biologicalactivity in the central region of the disc by increasing thepermeability or more specifically, the hydraulic and macromolecularpermeability and diffusivity of the outer region of the disc.

2. Description of the Related Art

Deleterious mechanical loading environments contribute to thedegradation of collagenous tissue in a variety of manners. For instance,fatigue is a weakening of a material due to repetitive applied stress.Fatigue failure is simply a failure where repetitive stresses haveweakened a material such that it fails below the original ultimatestress level. In bone, two processes—biological repair and fatigue—arein opposition, and repair generally dominates. In the intervertebraldisc, the prevalence of mechanical degradation of the posterior annulus(Osti 1992) suggests that fatigue is the dominant process. Active tissueresponse (adaptation, repair) does not play a strong role in the case ofmature intervertebral disc annular material. The intervertebral disc iscomprised of three parts: the nucleus pulposus (NP) or nucleus, theannulus fibrosus (AF) or annulus, and the cartilaginous endplates. Thecharacteristic of the inner annulus and outer nucleus blend with ongoingdegeneration, with the nucleus becoming more fibrous and decreasing inwater content. Similarly, the boundary between outer nucleus and innerannulus is known to fade and becomes indistinct with ongoingdegeneration. As a principally avascular structure, the disc relies ondiffusion for nutrition of its limited number of viable cells. Agerelated changes interfere with diffusion presumably contributing todeclining cell viability and biosynthetic function (Buckwalter et al.1993, Buckwalter 1995). Age related decline in numbers of cells and cellfunctionality compromises the ability of the cells to repair mechanicaldamage to the matrix. Regeneration of the matrix in the nucleusfollowing enzymatic degradation has been accomplished, albeitinconsistently (Deutman 1992). Regeneration of functional annularmaterial has not yet been realized.

Combined with this limited potential for repair or regeneration, studieshave shown that posterior intervertebral disc tissue is vulnerable todegradation and fatigue failure when subjected to non-traumatic,physiologic cyclic loads. Prior work has shown deterioration inelastic-plastic (Hedman 99) and viscoelastic (Hedman 00) materialproperties in posterior intervertebral disc tissue subjected to moderatephysiological cyclic loading. Cyclic load magnitudes of 30% of ultimatetensile strength produced significant deterioration of materialproperties with as little as 2000 cycles. Green (1993) investigated theultimate tensile strength and fatigue life of matched pairs of outerannulus specimens. They found that fatigue failure could occur in lessthan 10,000 cycles when the vertical tensile cyclic peak exceeded 45% ofthe ultimate tensile stress of the matched pair control. In addition,Panjabi et al (1996) found that single cycle sub-failure strains toanterior cruciate ligaments of the knee alter the elasticcharacteristics (load-deformation) of the ligament. Osti (1992) foundthat annular tears and fissures were predominantly found in theposterolateral regions of the discs. Adams (1982) demonstrated thepropensity of slightly degenerated discs to prolapse posteriorly whenhyperflexed and showed that fatigue failure might occur in lumbar discsas the outer posterior annulus is overstretched in the verticaldirection while severely loaded in flexion. Disc prolapse involvestearing and fissuring of the annulus fibrosus or outer portion of theintervertebral disc with migration and extrusion of central, nucleuspulposus materials. In an analytical study, interlaminar shear stresses,which can produce delaminations, have been found to be highest in theposterolateral regions of the disc (Goel 1995). These prior dataindicate: 1) the posterior disc and posterior longitudinal ligament areat risk of degenerative changes, and that 2) the mechanism ofdegeneration can involve flexion fatigue with possible fissuring,tearing and delamination.

A different type of mechanical degradation of collagenous tissue occursin scoliosis and other progressive spinal deformities. Scoliosis refersto an abnormal lateral, primarily, or other curvature or deformity ofthe spine, often of unknown origin. Progressive spinal deformities canalso occur subsequent to surgical bone removal, with or withoutaccompanying spinal instrumentation, such as in a neural decompressionprocedure or subsequent to vertebral failure. The bony vertebral failureitself may occur as a result of trauma or of age related osteoporosis orosteopenia. Kyphotic deformity (loss of outward concavity or increase inoutward convexity), in the lumbar spine also known as flat-backsyndrome, is a frequent sequela to spinal fusion or installation ofspinal instrumentation, especially in the case of a long, multi-level,surgical construct. Severe curvature and ongoing curve progression canlead to many other health disorders including but not limited tocompromised respiratory function. In addition, one's lifestyle can beadversely affected and a loss of cosmesis can result. A large segment ofthe population is affected by scoliosis, approximately 2% of women and0.5% of men. Over 80% of scoliosis is of no known origin (i.e.,idiopathic). Approximately 80% of idiopathic scoliosis develops in youngpubescent adults. The incidence of deformity increases with age.Existing conservative approaches to limit curve progression such asexternal bracing can be awkward or restricting, and are of disputedvalue. Surgical correction of severe curves can be intensive with a longrecovery period, require the need for post-operative bracing, and befraught with many other post-operative problems.

Another form of spinal deformity, spondylolisthesis commonly occurs inthe lower lumbar region of the spine. Spondylolisthesis involves theslippage of one vertebral level relative to an adjacent level.Progressive listhesis leads to sciatica and pain. Surgical interventionis an option to prevent progressive slip, especially when the slip hasreached a greater amount of slip displacement or slip angle. However,nonsurgical means of preventing a slip to progress to the point wheresurgery is indicated have not been available previously.

Current treatments for scoliosis and other progressive spinaldeformities consist of bracing and surgery. The purpose of orthopaedicbraces is to prevent increasing spinal deformity, not to correctexisting deformity. Braces are generally used in children with anexpected amount of skeletal growth remaining, who have curve magnitudesin the range of 25 to 40 degrees. External braces are routinely used asa standard of care. Yet there is controversy regarding the effectivenessof external bracing. The magnitude of forces delivered to the spinecorresponding to brace loads applied to the torso cannot be quantifieddirectly. Larger forces applied to the torso may also result in braceinduced pathologies to the tissues in contact with the brace. Somestudies suggest that braces are effective in halting curve progressionin about 80 percent of afflicted children. But because the option to donothing but observe curve progression is inappropriate, there is nogenerally accepted percentage of these curves that would stopprogressing on their own or due to other factors.

Naturally occurring collagen crosslinks play an important role instabilizing collagenous tissues and, in particular, the intervertebraldisc. Significantly higher quantities of reducible (newly formed)crosslinks have been found on the convex sides than on the concave sidesof scoliotic discs (Duance, et al. 1998). Similarly, Greve, et al.(1988) found a statistically increased amount of reducible crosslinks inscoliotic chicken discs at the same time that curvatures wereincreasing. This suggests that there is some form of natural,cell-mediated crosslink augmentation that occurs in response to theelevated tensile environment on the convex side of scoliotic discs.Greve also found that there were fewer reducible crosslinks at the veryearly stages of development in the cartilage of scoliotic chickens. Theyconcluded that differences in collagen crosslinking did not appear to becausative because there was not a smaller number of crosslinks at laterstages of development. In fact, later on, when the scoliotic curve wasprogressing, there were statistically significant greater numbers ofcollagen crosslinks, perhaps in response to the curvature. Although notthe conclusion of Greve, this can be interpreted as being a sufficientdepletion of crosslinks in the developmental process with long enoughduration to trigger the progression of scoliotic curvature that waslater mended by a cellular response that produced higher than normallevels of crosslinks. These studies suggest that the presence ofnaturally occurring collagen crosslinks may be critical to preventongoing degradation and for mechanical stability of intervertebral disctissue in scoliotic spines.

It is important to note that these studies did not quantify theintegrity or crosslink quantities associated with the elastin andelastic fiber network which also plays a role in the mechanicalintegrity of these collagenous materials. Some of the benefit ofcrosslinking of the principally collagenous tissues like theintervertebral disc may also be attributed to an effect on the elastinand elastic fiber network and other proteins (such as link proteins) inthese collagenous tissues. In the same way that intramolecular,intermolecular and interfibrillar crosslinks of collagen molecules andfibers benefit the tissue and joint mechanics, including resistance todegradation, tears and deformity, and increased permeability,intramolecular, intermolecular and interfibrillar crosslinks involvingelastin and the elastic fiber network could provide benefits to thetissue and joint mechanics and nutrition. In fact, the same reagentseffective at augmenting collagen crosslinking may also augmentcrosslinks involving the elastin and elastic fiber network, or othertissue proteins.

It is well documented that endogenous (naturally occurring—enzymaticallyderived and age increasing non-enzymatic) and exogenous collagencrosslinks increase the strength and stiffness of collagenous,load-supporting tissues (Wang 1994, Chachra 1996, Sung 1999a, Zeeman1999, Chen 2001). Sung (1999b) found that a naturally occurringcrosslinking agent, genipin, provided greater ultimate tensile strengthand toughness when compared with other crosslinking reagents. Genipinalso demonstrated significantly less cytotoxicity compared to other morecommonly used crosslinking agents. With regard to viscoelasticproperties, Lee (1989) found that aldehyde fixation reducedstress-relaxation and creep in bovine pericardium. Recently, naturallyoccurring collagen crosslinks were described as providing ‘sacrificialbonds’ that both protect tissue and dissipate energy (Thompson, et al.2001). To date, there is no known reference in the literature as to theability of exogenous crosslinks to decrease the viscoelasticcharacteristic of hysteresis or to increase the ability of thecollagenous tissue to store energy. A need therefore exists to findbiochemical methods that enhance the body's own efforts to stabilizediscs in scoliotic and other progressively deforming spines byincreasing collagen crosslinks.

Mechanical degradation of collagenous tissue can also occur if theenvironment for biological activity in the central region of the disc ispoor. Tissue engineering is a burgeoning field which aims to utilizecells, special proteins called cytokines and synthetic and nativematrices or scaffolds in the repair and regeneration of degraded,injured or otherwise failed tissues. With regard to the intervertebraldisc, biological solutions like tissue engineering are hindered by theharsh, hypoxic (oxygen deficient) avascular (very little if any directblood supply) environment of moderately degenerated intervertebraldiscs. The disc is known to receive nutrients and discard cell wasteproducts primarily by diurnal-cyclic pressure driven fluid flow anddiffusion through the annulus fibrosus and through the cartilaginousendplates that connect the disc to the bony, well vascularized, spinalvertebrae. The disc cartilaginous endplates lose permeability bycalcification while the disc itself becomes clogged up with anaccumulation of degraded matrix molecules and cell waste products. Thisloss of disc permeability effectively reduces the flow of nutrients tothe cells and the flow of waste products from the cells in the interiorcentral region of the disc, the nucleus pulposus. This loss of flow ofnutrition to the disc causes a loss of cell functionality, cellsenescence, and causes a fall in pH levels that further compromises cellfunction and may cause cell death (Buckwalter 1995, Horner and Urban2001). Homer and Urban showed that density of viable cells was regulatedby nutritional constraints such that a decline in glucose supply led toa decrease in viable cells. Boyd-White and Williams (1996) showed thatcrosslinking of basement membranes increased permeability of themembranes to macromolecules such as serum albumin, crosslinked albumin,and a series of fluorescein isothiocyanate dextrans of four differentmolecular sizes. It is herein suggested, then, that increasedcrosslinking of the annulus fibrosus and/or the endplates ofintervertebral discs, though very different and more complex collagenoustissues than basement membranes, would provide for increased flow ofglucose and other nutritional macromolecules to cells and waste productsfrom the cells in the interior region of the disc, thus improving theirviability.

Intervertebral disc herniation involves fissuring, rupture or tearing ofthe annulus fibrosus followed by displacement of the central portion ofthe disc posteriorly or posterolaterally through the torn tissue. Thedeformed or displaced disc protrusion can compress a nerve root and/orthe spinal cord. Clinical symptoms associated with herniated discinclude back pain and radiculopathy including leg pain, sciatica andmuscle weakness. Treatments for herniated disc commonly compriseexcision of the protruding disc segment and other tissues suspected tobe involved with nerve compression and pain. Prior to tearing throughthe outer annular fibers the disc can bulge posteriorly potentiallyapplying pressure to neural elements. Approximately a decade typicallyseparates the first, acute incidence of low back pain and the onset ofradicular symptoms. There is currently no treatment available to preventdegeneration, annular tearing, nucleus migration, herniation andsciatica.

Similarly, emerging nucleus augmentation or replacement technologiesrely on the integrity of a surgically weakened annulus fibrosus toprevent migration and extrusion or expulsion of implanted materials ordevices. These materials and devices are typically targeted for patientsin the early stages of disc degeneration (Galante I-III), where there isless degradation of the annulus fibrosus because of the reliance onannulus integrity for the success of these implants. However the annulusis typically compromised further in order to implant these materials anddevices to the central region of the disc. Clinical data at this timesuggests that implant migration and extrusion is one of the maincomplications to this type of treatment. High rates of extrusion havebeen reported for some nucleus replacements, 10% for the device with themost clinical experience and 20-33% for another. A need therefore existsfor a method for resisting annulus tearing after or at the same time ofimplantation of a nucleus augmentation or nucleus replacement devices.Normal physiological loading can displace, extrude or expulse devicesand materials implanted into the center, nucleus region of theintervertebral disc. Consequently, a treatment capable of improvingannulus tear resistance could be useful both to prevent eminent discprotrusions and as an adjunct to a disc augmentation or nucleusreplacement procedure.

To date, no treatments capable of reducing mechanical degradation tonative, non-denatured collagenous tissues currently exist. In fact, noother collagenous tissue fatigue inhibitors have been proposed. A needtherefore exists for a method for improving the resistance ofcollagenous tissues in the human body to fatigue and for otherwisereducing the mechanical degradation of human collagenous tissues, inparticular, the posterior annulus region of the intervertebral disc. Inaddition, a need exists to increase resistance to scoliotic curveprogression and other progressive spinal deformities by treatment ofappropriate regions on the tensile side (convex) of affected discs andto improve permeability, particularly the hydraulic and macromolecularpermeability and diffusivity of the outer region of the disc, but alsothroughout the disc annulus in whole or in part and the cartilaginousendplates of the disc, the flow of nutrition, such as glucose and othernutritional macromolecules, to cells in the annulus and in the centralportion of the disc, and the flow of waste products from the cells.

Spinal deformities following vertebral fractures including kyphoticdeformities following vertebral compression fractures are sometimestreated by injecting a cement-like material into the intravertebralspace (vertebroplasty) sometimes following a vertebral heightrestoration procedure to reduce the deformity (kyphoplasty). Acomplementary procedure to increase the tension band restraint byincreasing and improving elastic characteristics of the tensile side ofthe affected discs would also be beneficial in preventing the incidenceof deformity as well as the progression of the deformity. Improvement ofthe tension band characteristics in this way stabilizes the spinalcolumn and is a means of internal, natural bracing.

Additional advantages and novel features of this invention shall be setforth in part in the description that follows, and in part will becomeapparent to those skilled in the art upon examination of the followingspecification or may be learned by the practice of the invention. Theadvantages of the invention may be realized and attained by means of theinstrumentalities, combinations, compositions, methods, devices, andapplication trays particularly pointed out in the appended claims.

SUMMARY OF THE INVENTION

It is one object of the present invention to provide a method ofimproving the resistance of native, non-denatured collagenous tissues inthe human body to mechanical degradation by contacting the tissue withcrosslinking reagents.

It is another object of the present invention to provide a method ofcurtailing the progressive mechanical degradation of intervertebral disctissue by enhancing the body's own efforts to stabilize aging discs byincreasing collagen crosslinks.

It is another object of the present invention to provide a method thatuses crosslinking reagents with substantially less cytotoxicity comparedto common aldehyde fixation agents in order to facilitate direct contactof these reagents to tissues in the living human body.

It is another object of the present invention to increase thecrosslinking of non-denatured disc annular tissue by directly contactingliving human disc tissue with appropriate concentrations of a non-toxiccrosslinking reagent (or a mixture of crosslinking reagents) such asgenipin (a geniposide) or proanthrocyanidin (a bioflavonoid).

It is another object of the present invention to provide a treatmentmethod for minimally invasive delivery of the non-cytotoxic crosslinkingreagent such as injections directly into the select tissue using aneedle, for example into the convex side of discs involved in thecurvature or potential curvature of the spine, or placement of atime-release delivery system such as a carrier gel or ointment, or atreated membrane or patch directly into or onto the target tissue.

It is another object of the present invention to a composition composedof non-toxic crosslinking reagents that can be used as effective fatigueinhibitors.

In accordance with the present invention, there is provided a method fortreatment of tissues where a deleterious mechanical loading environmentcontributes to the degradation of the tissue. The deleterious mechanicalloading environment may consist of normal physiological repetitiveloading, otherwise known as fatigue or normal sustained or posturalloading known as creep, which is also typically repetitive in nature,and therefore a form of fatigue. The present invention provides a methodfor treatment of degenerated intervertebral discs to improve fatigueresistance. The present invention also provides non-toxic crosslinkingcompositions that are effective fatigue inhibitors.

The present invention uses non-cytotoxic crosslinking reagents such asgenipin or proanthocyanidin, a bioflavinoid, or a sugar such as riboseor threose, or byproducts of metabolism and advanced glycationendproducts (AGEs) such as glyoxal or methylglyoxyl or an enzyme such aslysyl oxidase (LO) enzyme (either in purified form or recombinant), ortransglutaminase (Tgase), or a LO or Tgase promotor, or an epoxy or acarbodiimide to improve the stability of intervertebral discs inscoliotic or other mechanically insufficient or potentially deforming ordeforming spines to eliminate or augment the need for external bracing.Preferably, the crosslinking reagent contains one of the followingranges of agent concentrations or a combination of agent concentrations:at least 0.001% (0.01 mg/ml) of human recombinant transglutaminase, atleast 0.01% (0.1 mg/ml) of purified animal liver transglutaminase, atleast 0.25% genipin, at least 0.1% proanthrocyanidin, at least 100 mMEDC, at least 100 mM ribose, at least 100 mM L-Threose, at least 50 mMmethylglyoxal, at least 50 mM glyoxal, at least 0.001% lysyl oxidase ina 0.1 M urea solution. In the case of non-enzymatic agents such asribose, L-Threose, methylglyoxal and glyoxal, the reagent willpreferably contain an oxidant such as hydrogen peroxide, or sodiumpercarbonate, or sodium borate, or an amino acid hydroperoxide, orperborate, or a buffer such as sodium bicarbonate or phosphate, or somecombination of oxidants and buffers. Further, the crosslinking reagentmay include a crosslinking agent in a carrier medium.

A method of improving the resistance of collagenous tissue to mechanicaldegradation in accordance with the present invention comprises the stepof contacting at least a portion of a collagenous tissue with aneffective amount of crosslinking reagent. The collagenous tissue to becontacted with the crosslinking reagent is preferably a portion of anintervertebral disc or similar fibrous collagenous tissue such as kneemeniscus. The contact between the tissue and the crosslinking reagent iseffected by injections directly into the select tissue using a needle.Alternatively, contact between the tissue and the crosslinking reagentis effected by placement of a time-release delivery system such as a gelor ointment, or a treated membrane or patch directly into or onto thetarget tissue. Contact may also be effected by, for instance, soaking orspraying.

It is another object of the present invention to provide biochemicalmethods that enhance the body's own efforts to stabilize discs inscoliotic and mechanically insufficient spines by increasing collagencrosslinks.

It is another object of the present invention to cause this stabilityenhancement by reducing the bending hysteresis (energy lost in acomplete loading-unloading cycle) which leaves an increased angle aftera deforming force is applied to the deformed joint of scoliotic ormechanically insufficient spines, that is increasing the “bounce-back”characteristics from a deformity-increasing load by injecting non-toxiccrosslinking reagents into the convex or tensile side of discs involvedin the scoliotic curve or potential or progressing deformity.

It is another object of the present invention to cause this stabilityenhancement by increasing the bending elastic energy storage and return(elasticity) of scoliotic or mechanically insufficient or potentiallydeforming or deforming spines by injecting non-toxic crosslinkingreagents into the convex side of discs involved in the potential orprogressing deformity curve.

It is another object of the present invention to cause this stabilityenhancement by increasing the bending stiffness (resistance to thedeformity-increasing bend) of scoliotic or mechanically insufficient orpotentially deforming or deforming spines by injecting non-toxiccrosslinking reagents into the convex side of discs involved in thepotential or progressing deformity curve.

The less energy lost in deformity-increasing bending, or the lesshysteresis in a bending cycle in the direction of increasing theexisting or potential deformity, means that a greater amount of energywas stored and can be recovered in the form of immediate recovery ofpre-bending shape. Greater hysteresis reflects a slower recovery of thepre-loaded shape and therefore a greater propensity for increasing thedeforming moments on the deformed joint (deforming moments increase withincreasing deformity) and, therefore, a greater propensity for increaseddeformity.

The appropriate locations for injection of the crosslinking reagent maybe determined using three-dimensional reconstructions of the affectedtissues as is possible by one skilled in the art, and combining thesereconstructions with an algorithm to recommend the optimum placement ofthese reagents so as to affect the greatest possible restraint ofpotential or progressive deformity or ongoing scoliotic curveprogression. These three-dimensional depictions of preferred locationsfor crosslinker application may best be created with custom computersoftware that incorporates any type of medical images of the patientthat are available, and may best be displayed on a computer drivendisplay device such as a lap-top computer or a devoted device.Additional, guidable, arthroscopic types of devices may be used, ordeveloped or modified, to facilitate application of the reagents toappropriate areas on the intervertebral discs or adjacent bony, capsularor ligamentous tissues.

It is another object of the present invention to increase thepermeability, particularly the hydraulic and macromolecular permeabilityand diffusivity, of the outer region of the intervertebral disc, theannulus fibrosus, and/or the cartilaginous endplates and by this improvethe fluid and solute and suspended particulate flux to improve the flowof nutrition, such as glucose and other nutritional macromolecules, tocells in the annulus and in the central region, or nucleus pulposus, ofan intervertebral disc, and the flow of waste products from the cells,by increasing collagen crosslinks.

It is another object of the present invention to increase the biologicalviability of cells in the central region of the intervertebral disc byincreasing collagen crosslinks.

The present invention then also relates to a new use of thenon-cytotoxic crosslinking reagent to improve the permeability of theouter regions of the intervertebral disc, possibly including theendplates, providing for an increased flux of fluids and solutes to andfrom the central region of the disc, thus improving the nutrition to thecells in this central region and the outflow of wasteproducts from thisregion. The collagenous tissue to be contacted with the crosslinkingreagent is preferably a portion of an intervertebral disc or similarfibrous collagenous tissue such as knee meniscus. These reagents arepreferably injected or otherwise applied to the majority of the outerannular regions of the intervertebral disc and to the endplates at thesuperior and inferior aspects of the disc. Additional, guidable,arthroscopic types of devices may be developed to facilitate applicationof the reagents to appropriate areas on the intervertebral discs.

It is another object of the present invention to increase the tearresistance of collagenous tissues such as the annulus fibrosus of theintervertebral disc and the knee meniscus, by increasing collagencrosslinks.

It is another object of the present invention to prevent expulsion orextrusion of disc nucleus materials or nucleus augmentation orreplacement materials or devices by increasing resistance to tissuefissuring and tearing.

In accordance with the present invention, there is provided a method fortreatment of tissues to increase resistance to tissue tearing. Thepresent invention provides a method for treatment of degeneratedintervertebral discs to resist disc herniation. The present inventionalso provides non-toxic crosslinking compositions that are effectivetearing inhibitors. The present invention provides a method forresisting the migration or expulsion or extrusion of materials ordevices implanted in the central nucleus region of intervertebral discs.The present invention also provides a method for treatment of kneemeniscus to hinder or prevent tissue tearing.

A method of improving the resistance of collagenous tissue to tearing inaccordance with the present invention comprises the step of contactingat least a portion of a collagenous tissue with an effective amount ofthe crosslinking reagent. The collagenous tissue to be contacted withthe crosslinking reagent is preferably a portion of an intervertebraldisc or similar fibrous collagenous tissue such as knee meniscus. Thecontact between the tissue and the crosslinking reagent is effected byinjections directly into the select tissue using a needle.Alternatively, contact between the tissue and the crosslinking reagentis effected by placement of a time-release delivery system such as a gelor ointment, or a treated membrane or patch directly into or onto thetarget tissue. Contact may also be effected by, for instance, soaking orspraying.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of relaxation (N) v. numbers of cycles showing theeffect of genipin crosslinking treatments (G1=0.033 g/mol, G2=0.33g/mol) on posterior intervertebral disc stress relaxation.

FIG. 2 is a graph of Brinnell's hardness index v. numbers of cyclesshowing the effect of genipin crosslinking treatments (G1=0.033 g/mol,G2=0.33 g/mol) on posterior intervertebral disc hardness or resistanceto penetration.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of improving the resistance ofcollagenous tissues in the human body to mechanical degradationcomprising the step of contacting at least a portion of a collagenoustissue with an effective amount of a crosslinking reagent. In oneembodiment of the present invention, the method of the present inventionalso provides a method of curtailing the progressive mechanicaldegradation of intervertebral disc tissue by enhancing the body's ownefforts to stabilize aging discs by increasing collagen crosslinks. Inthis embodiment, this mechanical degradation may be in response tophysiologic levels of repetitive loading.

The crosslinking reagent of the present invention is not particularlylimited. Any crosslinking reagent known to be substantiallynon-cytotoxic and to be an effective cross-linker of collagenousmaterial may be used. The crosslinking reagent is required to besubstantially non-cytotoxic in order to facilitate direct contact of thecrosslinking agent to tissues in the living human body. Preferably, thecrosslinking reagent exhibits substantially less cytotoxicity comparedto common aldehyde fixation agents. More preferably, a non-cytotoxiccrosslinking reagent is used.

Appropriate cytotoxicity testing will be used to verify the minimalcytotoxicity of candidate crosslinking reagents prior to use in humans.Tissue specific in vitro tests of cytotoxicity, of the standard formapplied to mouse connective tissue (F895-84(2001)e1 Standard Test Methodfor Agar Diffusion Cell Culture Screening for Cytotoxicity), or ChineseHamster Ovaries (ASTM E1262-88(1996) Standard Guide for Performance ofthe Chinese Hamster Ovary Cell/Hypoxanthine Guanine PhosphoribosylTransferase Gene Mutation Assay) preferably utilizing cell lines fromtissues approximating the fibrous and gelatinous tissues of theintervertebral disc, should be conducted to evaluate the level oftoxicity of any specific combination of crosslinking reagents known tohave minimal cytotoxicity. These in vitro tests should similarly befollowed by in vivo animal tests prior to use in humans.

The crosslinking reagent includes at least one crosslinking agent. Thecrosslinking agent chosen in accordance with the present invention is aneffective cross-linker of collagenous material. When used in across-linking reagent, an effective crosslinker is one that increasesthe number of crosslinks in the collagenous tissue when the crosslinkeris brought into contact with a portion of the collagenous tissue. Aneffective crosslinker improves the fatigue resistance of the treatedtissue, reduces material property degradation resulting from repetitivephysiologic loading, increases resistance to tissue tearing, resistsprogressive deformity, increases hydraulic permeability of the tissue,or reduces the increase of viscoelastic properties of the treated tissuedue to fatigue loading. Likewise, an effective crosslinker may reducethe decrease in elastic-plastic properties due to fatigue loading of thetreated tissue.

In accordance with the invention, this method would utilize specificformulations of crosslinking reagents with substantially lesscytotoxicity compared to common aldehyde fixation agents in order tofacilitate direct contact of these reagents to tissues in the livinghuman body. Bioflavinoids and geniposides have been shown to beeffective crosslinkers with minimal cytotoxicity. Similarly, sugar(e.g., ribose or threose) solutions and byproducts of metabolism andadvanced glycation endproducts (AGEs) such as glyoxal or methylglyoxylhave been shown to increase the number of non-enzymatic glycationproduced crosslinks (naturally produced crosslinks, pentosidine is oneexample). Lysyl oxidase is the naturally produced enzyme involved in theformation of immature and mature endogenous (naturally occurring)collagen crosslinks. The method used to increase the crosslinking ofdisc annular tissue may include directly contacting living human disctissue with appropriate concentrations of minimally-cytotoxiccrosslinking reagents such as genipin or proanthocyanidin, abioflavinoid, or a sugar such as ribose or threose, or byproducts ofmetabolism and advanced glycation endproducts (AGEs) such as glyoxal ormethylglyoxyl or an enzyme such as lysyl oxidase (LO) enzyme (either inpurified form or recombinant), or transglutaminase (Tgase), or a LO orTgase promotor, or an epoxy or a carbodiimide. Preferably, thecrosslinking reagent contains one of the following ranges of agentconcentrations or a combination of agent concentrations: at least 0.001%(0.01 mg/ml) of human recombinant transglutaminase, at least 0.01% (0.1mg/ml) of purified animal liver transglutaminase, at least 0.25%genipin, at least 0.1% proanthrocyanidin, at least 100 mM EDC, at least100 mM ribose, at least 100 mM L-Threose, at least 50 mM methylglyoxal,at least 50 mM glyoxal, at least 0.001% lysyl oxidase in a 0.1 M ureasolution. In the case of non-enzymatic agents such as ribose, L-Threose,methylglyoxal and glyoxal, the reagent will preferably contain anoxidant such as hydrogen peroxide, or sodium percarbonate, or sodiumborate, or an amino acid hydroperoxide, or perborate, or a buffer suchas sodium bicarbonate or phosphate, or some combination of oxidants andbuffers. More than one crosslinking agent may be used.

The crosslinking reagent may include a carrier medium in addition to thecrosslinking agent. The crosslinking agent may be dissolved or suspendedin the carrier medium to form the crosslinking reagent. In oneembodiment, a crosslinking agent is dissolved in a non-cytotoxic andbiocompatible carrier medium. The carrier medium is required to besubstantially non-cytotoxic in order to mediate the contact of thecrosslinking agent to tissues in the living human body withoutsubstantial damage to the tissue or surrounding tissue. Preferably, thecarrier medium chosen is water, and more preferably, a saline solution.Preferably, the pH of the carrier medium is adjusted to be the same orsimilar to the healthy tissue environment. Even more preferably, thecarrier medium is buffered. In one embodiment of the present invention,the carrier medium is a phosphate buffered saline (PBS).

When the crosslinking agent is dissolved in a carrier medium, theconcentration of the crosslinking agent in the carrier medium is notparticularly limited. The concentration may be in any amount effectiveto increase the crosslinking of the tissue while at the same timeremaining substantially noncytotoxic.

In accordance with the present invention, the crosslinking reagent isbrought into contact with a portion of a native, non-denaturedcollagenous tissue. As used herein, collagenous tissue is defined to bea structural or load supporting tissue in the body comprised of asubstantial amount of collagen. Examples would include intervertebraldisc, articular cartilage, meniscus, fibrocartilage, ligament, tendon,bone, and skin. In general, the portion of the collagenous tissue to bebrought into contact with the crosslinking reagent is the portion of thetissue that is subject to loading. Further, where at least somedegradation of the collagenous tissue has occurred, the portion of thetissue to be contacted with the crosslinking reagent is at least theportion of the tissue that has been degraded or deformed. Preferably,the entire portion that is subject to loading or the entire portion thatis degraded or deformed is contacted with the crosslinking reagent.Further, the tissue adjacent the portion of collagenous tissue subjectto the loading may also be contacted with the crosslinking reagent.

The collagenous tissues that are particularly susceptible for use inaccordance with the present invention include intervertebral discs andarticular cartilage or fibrocartilage such as knee meniscus. Where thecollagenous tissue is an intevertebral disc, the portion of theintervertebral disc that is preferably contacted by the crosslinkingreagent is the posterior and posterolateral annulus fibrosis.

The selected portion of the collagenous tissue must be contacted with aneffective amount of the non-toxic crosslinking reagent. An “effectiveamount” is an amount of crosslinking reagent sufficient to have amechanical effect on the portion of the tissue treated. Specifically, an“effective amount” of the crosslinking reagent is an amount sufficientto improve the fatigue resistance of the treated tissue, reduce materialproperty degradation resulting from repetitive physiologic loading,increases resistance to tissue tearing, increases resistance todeforming forces, increases hydraulic permeability of the tissue, orreduce the increase of viscoelastic properties of the treated tissue dueto fatigue loading, or reduce the decrease of elastic-plastic propertiesof the treated tissue due to fatigue loading. An effective amount may bedetermined in accordance with the viscoelastic testing, tear testing,deformity resistance testing, hydraulic permeability testing, and/or theelastic-plastic testing described herein with respect to Examples 1, 2,3, 4 and 5.

The method of the present invention includes contacting at least aportion of the collagenous tissue with an effective amount of thecrosslinking reagent. The contact may be effected in a number of ways.Preferably, the contacting of collagenous tissue is effected by a meansfor minimally invasive delivery of the non-cytotoxic crosslinkingreagent. Preferably, the contact between the tissue and the crosslinkingreagent is effected by injections directly into the select tissue usinga needle. Preferably, the contact between the tissue and thecrosslinking reagent is effected by injections from a single or minimumnumber of injection locations. Preferably, an amount of crosslinkingsolution is injected directly into the targeted tissue using a needleand a syringe. Preferably, a sufficient number of injections are madealong the portion of the tissue to be treated so that complete coverageof the portion of the collagenous tissue to be treated is achieved.

Alternatively, contact between the tissue and the crosslinking reagentis effected by placement of a time-release delivery system directly intoor onto the target tissue. One time-released delivery system that may beused is a treated membrane or patch. A reagent-containing patch may berolled into a cylinder and inserted percutaneously through a cannula tothe tissue sight, unrolled and using a biological adhesive or resorbablefixation device (sutures or tacks) be attached to the periphery of thetargeted tissue.

Another time-released delivery system that may be used is a gel orointment. A gel or ointment is a degradable, viscous carrier that may beapplied to the exterior of the targeted tissue.

Contact also may be effected by soaking or spraying, such asintra-capsular soaking or spraying, in which an amount of crosslinkingsolutions could be injected into a capsular or synovial pouch.

A form of mechanical degradation of load supporting collagenous tissuesincludes tearing of the tissues. In a second embodiment, the presentinvention relates to methods and devices for the treatment of fibrouscollagenous tissues and surrounding tissues by directly contacting theselect tissues with a crosslinking reagent to improve the resistance totearing of the tissue. The collagenous tissues that are particularlysusceptible to tearing include intervertebral discs and articularcartilage or fibrocartilage such as knee meniscus. Where the collagenoustissue is an intervertebral disc, the portion of the intervertebral discthat is preferably contacted by the crosslinking reagent is theposterior and posterolateral annulus fibrosis. Where the treated tissuesinclude the intervertebral disc, the present invention also relates tomethods and devices for prevention of disc herniation. The presentinvention could be used in a conservative approach to prevent tearing ofdisc tissue, in particular, radial tearing of the annulus fibrosusleading to expulsion of nucleus pulposus materials.

One aspect of this embodiment provides a method of improving the tearresistance of native intervertebral disc tissue prior to a discherniation by contacting the tissue with a non-toxic crosslinkingreagent. This embodiment also provides a method of improving tearresistance where a herniation has already occurred by contacting thetissue with non-toxic crosslinking reagents. Another aspect of thisembodiment provides a method of improving the tear resistance of nativeknee meniscus tissue subsequent to a partial tearing of the meniscus.This embodiment also provides a method of improving tear resistance ofnative knee meniscus tissues prior to tearing of the meniscus.

In this embodiment, an effective amount of crosslinking reagent is anamount that creates crosslinks in the target tissue, preferably inregions of the tissue where the majority of tissue tearing is known tooccur. In the case of intervertebral disc tissues, the treatment ispreferably on the posterior and posterolateral regions of the annulusfibrosus such that the resistance to tearing is increased for theprevention of disc herniation. In the case of knee meniscus tissues, thetissue is preferably treated in the region of existing partial tears andto surrounding meniscal tissues. The knee meniscal tissue is alsopreferably treated in its entirety. If only the medial meniscus or onlythe lateral meniscus is affected, both the medial and lateral meniscuscan be treated. If the meniscal tissues in one knee is affected, thecrosslinking treatment could also be made on the meniscal tissues in thecontralateral knee.

Preferably, a method according to this embodiment uses a minimallyinvasive delivery of the non-cytotoxic crosslinking reagents, such as aseries of injections, to the affected discs or knee meniscus orcartilaginous or bony or capsular or ligamentous tissues in order tocontact the appropriate tissue with appropriate concentrations ofnon-toxic crosslinking reagents. This aspect of the present invention isused in a conservative approach to prevent ongoing tearing anddegeneration of these tissues and in the case of the intervertebraldisc, to prevent subsequent herniations of the disc.

Preferably, a treatment method according to this embodiment incorporatesa means for minimally invasive delivery of the non-cytotoxiccrosslinking reagent such as placement of a time-release delivery systemsuch as an imbedded pellet or time release capsule, or a treatedmembrane or patch directly into or onto the target tissue. Additional,guidable, arthroscopic-types of devices may be developed to facilitateapplication of the reagents to appropriate areas on the intervertebraldiscs or knee meniscus or adjacent cartilaginous, bony, capsular orligamentous tissues. This aspect of the present invention is used in aconservative approach to prevent ongoing tearing and degeneration ofthese tissues and in the case of the intervertebral disc, to preventsubsequent herniations of the disc.

In a third embodiment, the present invention relates to methods anddevices for the treatment of intervertebral discs subsequent to or incombination with a nucleus replacement or nucleus augmentation typeprocedure, and to prevention of extrusion or expulsion of the implantedmaterials and/or devices. Nucleus pulposus replacement or augmentationtechnologies rely on the integrity of a surgically weakened annulusfibrosus to prevent migration and extrusion or expulsion of implantedmaterials or devices. The present invention could be used for resistingannulus tearing after or at the same time of implantation of a nucleusaugmentation or nucleus replacement devices. The present inventionprovides methods and devices such that an injectable nucleus replacementor augmentation materials could be combined with crosslinking reagentsto prevent extrusion of the implanted materials. This embodiment wouldinvolve delivery of the crosslinking reagents to the central region ofthe disc and would create an inside-out progression of crosslinking,with the more central annulus tissues being in direct contact with thecrosslinking reagents. Alternatively, the present invention provides fora separate crosslinking treatment of the annulus performed atapproximately the same time as a nucleus replacement or augmentationprocedure. Alternatively, the present invention provides for acrosslinking treatment of the annulus performed subsequent to a nucleusreplacement or augmentation procedure. Also, this invention provides forthe treatment of the annulus preceding the implantation of the nucleusmaterials or devices.

In this embodiment, an effective amount of crosslinking reagent is anamount that creates crosslinks in the target tissue, preferably on allof the annulus surrounding a nucleus replacement implant material ordevice, also preferably on the region surrounding the defect formed bysurgical introduction of the implant, also preferably on the surroundingtissues including the cartilaginous vertebral endplates and capsular andligamentous tissues, such that the annulus resistance to tearing isincreased.

Preferably, a method according to this embodiment uses a minimallyinvasive delivery of the non-cytotoxic crosslinking reagents. Aminimally invasive delivery includes a series of injections, to thetarget tissues of or adjacent to discs which have received or arereceiving or will receive a nucleus replacement or augmentationprocedure, in order to contact the appropriate tissue with appropriateconcentrations of non-toxic crosslinking reagents. This aspect of thepresent invention is used in a conservative approach to prevent furthertearing of the annulus prior to or following implantation of a nucleusreplacement device or materials, and to prevent migration or expulsionor extrusion of implant materials or devices.

Another minimally invasive delivery includes placement of a time-releasedelivery system such as an imbedded pellet or time release capsule, or atreated membrane or patch directly into or onto the target tissue.Additional, guidable, arthroscopic-types of devices may be developed tofacilitate application of the reagents to appropriate areas on theintervertebral discs or adjacent cartilaginous, bony, capsular orligamentous tissues. This aspect of the present invention is used in aconservative approach to prevent further tearing of the annulus prior toor following implantation of a nucleus replacement device or materials,and to prevent migration or expulsion or extrusion of implant materialsor devices.

A fourth embodiment of the present invention provides methods anddevices for enhancing the body's own efforts to stabilize discs inscoliotic spines or other mechanically insufficient or potentiallydeforming or deforming spines such as listhetic spines, (which containsat least one partially slipped disc), those following a neuraldecompression procedure such as a laminectomy or subsequent toinstallation of spinal instrumentation, by increasing collagencrosslinks. A form of mechanical degradation to intervertebral discsoccurs as a part of progressive curvature of the spine. For example,spinal curve progression in scoliosis involves increased unloadedcurvature of segments of the spine. With this increased curvature thereis an associated increase of gravity-induced bending moments on thespine, acting to increase the curvature of these already affectedjoints. Although it may also be considered as a sustained or static typeof load, with a period of loading equal to the period of uprightactivity during any given day, the “repetitive” or fatigue loadingassociated with scoliosis curve progression would be comprised of thedaily gravitational loads and passive and active muscle and connectivetissue actuated loads and their effective moments applied to the spinalcolumn over the course of many days. With increasing deformity, thedeforming moments are increased as the “moment arm”—the distance throughwhich the applied forces generate moments—increases. The fundamentalrationale behind scoliotic bracing, and bracing for other spinaldeformities is to resist these deforming forces and moments, affectingthe loading environment of the cells in the bones and connective tissue,and to resist curve progression. The present invention could be used ina conservative approach to prevent ongoing curvature of spines and as anadjunct to corrective surgery to stabilize the remaining discs againstloss of correction. It could be used alone or with external bracing.

One aspect of this embodiment provides a method of improving thestability of intervertebral disc tissue in scoliotic or othermechanically insufficient or potentially deforming or deforming spines,such as spondilolisthesis (“slipped discs”), aiding the cell's effortsto increase collagen crosslinks on the tensile (convex) side of thecurves and slips, by contacting the tissue with one or more of thenon-toxic crosslinking reagents.

In this embodiment, an effective amount of crosslinking reagent is anamount that creates crosslinks in the target tissue, preferably on theconvex side of discs at or near the apex or apexes of a spinal curve orof a potential spinal curve, such that at least one of the followingeffects are achieved: deformity-increasing bending hysteresis isdecreased, elastic energy storage and return is increased, and thedeformity-increasing bending stiffness is increased.

Preferably, a method according to this embodiment uses a minimallyinvasive delivery of the non-cytotoxic crosslinking reagents, such as aseries of injections, to the tensile (convex) sides of affected discsand adjacent bones, capsular or ligamentous tissues in order to contactthe appropriate tissue with appropriate concentrations of non-toxiccrosslinking reagents. The appropriate locations for injection aredetermined using three-dimensional reconstructions of the affectedtissues as is possible existing technology, and combining thesereconstructions with an algorithm to recommend the optimum placement ofthese reagents so as to affect the greatest possible restraint ofongoing spinal curve progression. These three-dimensional depictions ofpreferred locations for crosslinker application may best be created withcustom computer software that incorporates medical images of thepatient, and are preferably displayed on a computer driven displaydevice such as a lap-top computer or a devoted device. This aspect ofthe present invention is used in a conservative approach to preventongoing curvature of scoliotic and other progressively deforming spinesand as an adjunct to corrective surgery to stabilize the remaining discsagainst loss of correction. It is used alone or with external bracing.

Preferably, a treatment method according to this embodiment incorporatesa means for minimally invasive delivery of the non-cytotoxiccrosslinking reagent such as placement of a time-release delivery systemsuch as an imbedded pellet or time release capsule, or a treatedmembrane or patch directly into or onto the target tissue. Additional,guidable, arthroscopic-types of devices may be developed to facilitateapplication of the reagents to appropriate areas on the intervertebraldiscs or adjacent bony, capsular or ligamentous tissues. This aspect ofthe present invention is used in a conservative approach to preventongoing curvature of spines and as an adjunct to corrective surgery tostabilize the remaining discs against loss of correction. It is usedalone or with external bracing.

Decreased diffusion into the central portion of the intervertebral discis strongly related to the loss of cell function in the disc and discdegeneration. This loss of diffusion capabilities affects both thecartilaginous endplates of the disc (above and below) and the outerregion of the disc, the annulus fibrosus. A fifth embodiment of thepresent invention provides methods and devices for increasing loadsupporting collagenous tissue permeability and the flow of nutrition byincreasing collagen crosslinks by using one or more of the crosslinkingreagents. The present invention increases changes in the hydration ofvarious regions of an intervertebral disc in a way that demonstrates anincreased fluid flow into and out of the central region, or nucleuspulposus, of the intervertebral disc afforded by increased crosslinkingof the outer region of the disc, the annulus fibrosus and/or thecartilaginous endplates. The changes effected by crosslinking increasethe hydraulic permeability of the outer regions of the disc and increasesolute transport to and from the central regions of the disc. Also, thepresent invention increases hydraulic permeability of the outer regionsof the knee meniscus tissues and increases solute transport to and fromthe inner regions of the knee meniscus.

One aspect of this embodiment provides a method to increase thepermeability of the outer region of the intervertebral disc, the annulusfibrosus and/or the cartilaginous endplates, and by this improve thefluid flux to and from the central region, or nucleus pulposus, of anintervertebral disc by increasing collagen crosslinks.

A second aspect of this embodiment provides a method to increase theouter disc permeability and increase fluid flux to the central region ofthe disc to increase the flow of nutrients to the cells in the centralregion, while also increasing the flow of cell waste products anddegraded matrix molecules from the central region of the disc, byincreasing collagen crosslinks.

A third aspect of this embodiment provides a method to increase thebiological viability of cells or the effectiveness of cell stimulatingagents such as cytokines and growth factors in the central region of theintervertebral disc by increasing collagen crosslinks.

This embodiment provides a method for improving flow of nutrients to thecentral region of the intervertebral disc while also improving outflowof waste products from this central region. This improvement of flow isbrought about by increased permeability of the outer region of the discproduced by application of crosslinking reagents to this outer region.This embodiment also provides a method for improving flow of nutrientsto the central region of the knee meniscus while also improving outflowof waste products from this central region.

Methods according to this embodiment use a minimally invasive deliveryof the non-cytotoxic crosslinking reagents, such as a series ofinjections, or the placement of a time-release delivery system such asan imbedded pellet or time release capsule, or a treated membrane orpatch directly into or onto the target tissue. Additional, guidable,arthroscopic-types of devices may be developed to facilitate applicationof the reagents to appropriate target areas. These delivery methods areused in a conservative approach to increase the fluid flow, solutetransport, nutrient supply, and waste removal to the central region ofthe disc or meniscus by crosslinking treatment of the outer region, orannulus of the disc or meniscus. These delivery methods function as anessential adjunct to tissue engineering treatments of the intervertebraldisc to improve the viability of the implanted or otherwise treatedcells and to effect an increase in the biological activity of the discor meniscus. Tissue engineering treatments and cell or cytokine basedmethods may include any of the following: implantation of stem cells ofany derivation (autogenous or autologous, embryonic or non-embryonic,muscle derived, adipose derived, etc.), gene-therapy delivery of growthfactors, implantation of matrices with attached growth factors, directapplication of growth factors, implantation of transplanted tissues orcells, implantation of xenograft tissues or cells, to promote increasedbiological activity in the disc or meniscus. In addition, these deliverymethods will be used where no tissue engineering type of treatment isapplied with the aim to increase diffusion to the central region of thedisc, the nucleus pulposus, or to the central region of the kneemeniscus.

It should be noted that the methods and compositions treated herein arenot required to permanently improve the resistance of collagenoustissues in the human body to mechanical degradation, or to permanentlyincrease resistance to tissue tearing, or to permanently increase tissuepermeability, or to permanently increase resistance to deformity.Assuming that a person experiences 2 to 20 upright, forward flexionbends per day, the increased resistance to fatigue, deformity andtearing and the increase in permeability associated with contact of thecollagenous tissue with the crosslinking reagent, may, over the courseof time, decrease. Preferably, however, the increased resistance tofatigue, deformity and tearing and the increased permeability lasts fora period of several months to several years without physiologicmechanical degradation. Under such circumstance, the described treatmentcan be repeated at the time periods sufficient to maintain an increasedresistance to fatigue, deformity and tearing and increased permeability.Using the assumption identified above, the contacting may be repeatedperiodically to maintain the increased resistance to fatigue, deformityand tearing and increased permeability. For some treatment, the timebetween contacting is estimated to correspond to approximately 1 yearfor some individuals. Therefore, with either a single treatment or withrepeated injections/treatments, the method of the present inventionminimizes mechanical degradation of the collagenous tissue over anextended period of time.

Another aspect of the present invention relates to using theaforementioned crosslinking agents as a device or “reagent andapplication tray” for improving the stabilization of intervertebraldiscs, for improving the resistance of collagenous tissue to mechanicaldegradation, for increasing the permeability of the intervertebral disc,for improving the fluid flux to and from the intervertebral disc, andfor increasing the biological viability of cells in the intervertebraldisc.

The “reagent and application tray” is sterile and contained within asterile package. All of the necessary and appropriate and pre-measuredreagents, solvents and disposable delivery devices are packaged togetherin an external package that contains a suitable wrapped sterile “reagentand application tray”. This sterile tray containing the reagents,solvents, and delivery devices is contained in a plastic enclosure thatis sterile on the inside surface. This tray will be made availableseparate from the computer hardware and software package needed tosuggest appropriate application positions.

EXAMPLES 1 AND 2

Thirty-three lumbar intervertebral joints were obtained from tenfour-month-old calf spines. The intervertebral joints were arbitrarilydivided into 3 groups: untreated controls-12 specimens, Genipintreatment 1 (G1)-6 specimens, and Genipin treatment 2 (G2)-13 specimens.The G1 treatment involved 72 hours of soaking the whole specimen in PBSwith a 0.033% concentration of Genipin. Similarly the G2 treatmentinvolved 72 hours of soaking whole specimens in PBS with 0.33%concentration of Genipin. 0.33% Genipin in PBS is produced by dilutionof 50 ml of 10.times. PBS (Phosphate Buffered Saline) with distilledwater by a factor of 10 to give 500 ml (500 gm) of PBS and mixing in1.65 grams of genipin to produce the 0.33% (wt %, gm/gm) solution.Previous testing with pericardium and tendon tissue samples demonstratedthe reduction of tissue swelling (osmotic influx of water into thetissue) resulting from crosslinking the tissue. Some controls were notsubjected to soaking prior to fatigue testing. Others were soaked in asaline solution for 72 hours. Water mass loss experiments were conductedto establish the equivalency of outer annulus hydration between thegenipin soaked and 0.9% saline soaked controls. The selection oftreatments was randomized by spine and level. The vertebral ends of thespecimens were then potted in polyurethane to facilitate mechanicaltesting.

Indentation testing and compression/flexion fatigue cycling were carriedout in the sequence presented in Table 1. TABLE 1 Experimental protocolMeasurement Sequence Measurement Location 1 Stress Relaxation Center ofthe Posterior Annulus 2 Hardness Center of the Posterior Annulus 3000Compression/Flexion Fatigue Cycles 3 Stress Relaxation 4 mm Lateral toCenter 4 Hardness Center of the Posterior Annulus Additional 3000Compression/Flexion Fatigue cycles 5 Stress Relaxation 4 mm Lateral toCenter 6 Hardness Center of the Posterior Annulus

At the prescribed points in the loading regimen, indentation testing wasused to find viscoelastic properties as follows. Stress relaxation datawas gathered by ramp loading the 3 mm diameter hemi-spherical indenterto 10 N and subsequently holding that displacement for 60 s, whilerecording the resulting decrease in stress, referred to as the stressrelaxation. Indentation testing was also utilized to determineelastic-plastic properties by calculating a hardness index (resistanceto indentation) from ramp loading data. Prior to recording hardnessmeasurements, the tissue is repeatedly indented 10 times (60 s/cycle, tothe displacement at an initial 10 N load).

This test protocol is based on two principles. First, viscoelasticeffects asymptotically decrease with repeated loading. Secondly,hardness measurements are sensitive to the loading history of thetissue. However this effect becomes negligible following 10 loadingcycles. In order to minimize these effects, viscoelastic data (stressrelaxation) was collected from tissue that had not previously beenindented. Alternately, elastic-plastic data (hardness) was collectedfrom tissue that had been repeatedly loaded (preconditioned). In thiscase, repetitive indentation was intended to reduce the undesiredeffects of the changing viscoelastic properties, namely lack ofrepeatability, on hardness measurements. These testing procedures werederived from several preliminary experiments on the repeatability of themeasurements with variations of loading history and location.

Following initial indentation testing, the specimen was loadedrepetitively in flexion-compression at 200 N for 3000 cycles at a rateof 0.25 Hz. The load was applied perpendicularly to the transverseplane, 40 mm anterior to the mid-point of the specimen in the transverseplane. A second set of indentation testing data is then collectedfollowing fatigue cycling. This procedure was followed for two fatigueloading cycles. During all testing, the specimens were wrapped in salinewetted gauze to maintain their moisture content. Fatigue cycling andnon-destructive indentation testing were carried out on an MTS 858.02biaxial, table-top, 10 kN capacity servo-hydraulic materials teststation (MTS, Eden Prairie, Minn.), with the MTS Test Star dataacquisition system. Several statistical measures were calculated toevaluate the significance of the results. A nested two-way analysis ofvariance (ANOVA) was utilized to confirm effects due to treatment andnumber of fatigue cycles. Due to the non-parametric nature of the data,the Mann-Whitney non-parametric rank-sum test was used to assess thenull hypotheses that the treatment did not affect: 1) the pre-cyclingmechanical parameters of the tissue, or 2) the amount of change(degradation) in elastic-plastic and viscoelastic mechanical parametersdue to fatigue loading. The confidence level for statisticalsignificance was set at p<0.05.

Nested two-way ANOVA analysis determined that both viscoelastic(relaxation) and elastic-plastic (hardness) mechanical parameters wereindependently affected by fatigue cycling and by treatment type. Thesestatistical results are presented in Table 2.

The relaxation test results are presented graphically in FIG. 1.

There was an initial shift downward of the relaxation curve caused bythe crosslinking treatment. This would represent a beneficial effect ashigher stress relaxation would be associated with more severely degradedtissue (Lee 1989). The initial pre-fatigue relaxation of the G1 and G2treatment groups were 26% and 19% less than (p=0.009 and p=0.026) thepre-fatigue relaxation of the controls respectively. There was alsodramatic improvement in fatigue resistance as demonstrated by the changein relaxation after 6000 non-traumatic loading cycles. The change inrelaxation due to 6000 fatigue cycles for the G2 treated discs was lessthan a third of the change in the controls (p=0.044). However, thelesser concentration of Genepin did not bring about the same improvementin fatigue resistance.

The hardness test results are presented graphically in FIG. 2. There isan initial shift upward of the hardness data caused by the G2crosslinking treatment. This would represent a beneficial effect as lossof hardness would signal a loss of structural integrity in the tissue.The initial pre-fatigue hardness of the G2 treatment group was 17%greater than that of the control group (p=0.026). However thisbeneficial effect appears to have eroded prior to 3000 fatigue cyclesand the change in hardness between 3000 and 6000 cycles is essentiallythe same for the two groups (G2=−0.94, Control=−1.01). TABLE 2 Resultsof nested two-way ANOVA analysis Material Property Factor F-ValueProbability Stress Relaxation Treatment 16.060 1.085E−06 Fatigue Cycling9.676 2.500E−03 Interaction 1.402 2.515E−01 Hardness Treatment 20.0236.405E−08 Fatigue Cycling 5.898 1.710E−02 Interaction 4.228 1.760E−02

The data presented above quantifies the elastic and viscoelasticmechanical degradation of intervertebral disc tissue due-to repetitive,non-traumatic loading. The results of these experiments establish thatnon-toxic crosslinking reagents reduce the fatigue-related degradationof material properties in a collagenous tissue—namely the intervertebraldisc. More than a three-fold reduction in viscoelastic degradation wasbrought about by soaking the calf disc tissue in 0.33 g/molconcentration of genipin. The tested formulation was unable to sustainan improvement in the elastic mechanical properties (hardness) to 3000test cycles.

Accurately estimating the length of time it would take an average personto experience a comparable amount of wear and tear on their spinal discsis difficult. Certainly, in addition to the mechanical degradationimposed by the described testing, there is theadded—“natural”—degradation of these dead tissues due to the testingenvironment. The non-loaded controls showed this “natural” degradationof material properties to be insignificant. Measures were taken tominimize this natural degradation by keeping the specimens moistthroughout the testing and by accelerating the loading frequency. At thesame time, loading frequency was kept within physiologic limits toprevent tissue overheating. It should be noted that these measuresconstitute standard protocol for in vitro mechanical testing ofcadaveric tissues. Assuming that a person experiences 2 to 20 upright,forward flexion bends per day, these data roughly correspond to severalmonths to several years of physiologic mechanical degradation.

The described treatment could be repeated at the time periodsrepresented by, for instance, 3000 fatigue cycles at this loadmagnitude. Using the assumption identified above, this number of cyclesmay be estimated to correspond to approximately 1 year for someindividuals. Therefore, with either a single treatment or with repeatedinjections/treatments, an individual may be able to minimize mechanicaldegradation of their intervertebral discs over an extended period oftime. Another option would involve a time-release delivery system suchas a directly applied treated patch, a gel or ointment.

EXAMPLES 3 AND 3a

Experiments were conducted to evaluate the efficacy of applyingdifferent formulations of crosslinking reagents with known minimalcytotoxicity unilaterally to intervertebral disc annular tissue in orderto affect the lateral bending stability of the tissue compared topre-treatment.

Experiments utilized 5 calf spine segments, each segment comprised of 3lumbar intervertebral joints (motion segments), four vertebrae and theintervening 3 discs. The pedicles were cut and the posterior processesremoved. The segments were randomly divided into a 0.33% by weightgenipin crosslinked group, a 0.5% genipin group, a 0.66% genipin group,and a 0.66% genipin plus 0.1% proanthocyanidin group. Each groupconsisted of one 3 motion segment specimen. Each pre-treated spineserved as its own control. Repeated testing was performed on someuntreated and treated specimens to determine repeatability of themeasurements. Additional appropriate concentrations and combinations ofknown minimally cytotoxic crosslinking reagents will be chosen based onthe documented cytotoxicity of a particular tissue. In this regard it isexpected that sugar solutions (such as ribose and 1-threose), byproductsof metabolism (such as methylglyoxal and glyoxal) and naturallyoccurring enzymes (such as lysyl oxidase and transglutaminase) will beessentially non-cytotoxic. Similar testing will be conducted onfresh-non-frozen animal tissue with appropriate sterilization proceduresand antibiotics to prevent tissue degradation. Sugar solutions will beinjected unilaterally into fresh intervertebral discs to inducenon-enzymatic glycation crosslinks over a period of sterile incubation.

Four-point lateral bending tests were conducted using an MTS 858materials testing system with custom fixtures while load anddisplacement were recorded digitally. First the specimens were cleanedof muscle and other non-load supporting tissues, and then the terminalvertebrae were potted in polyurethane to half their height in squaremolds. The potted spine segments are then placed on the bottom 2 rollerssuch that the lateral sides of the spines were positioned in a verticalplane. The bending load was actuated by 2 upper rollers in contact withthe central two vertebrae of the segment. Care was taken to ensure thatthe pre- and post-treatment positioning of the specimens on the rollerswas similar. As an attribute of 4-point bending, the central region ofthe test specimen, including the central disc between the 2 upperrollers, has an evenly distributed shear load and bending moment. A rampload to 100 N (0.5 mm/s) was applied in right and left lateral bendingto each spine both prior to treatment and after crosslinking treatment.

The crosslinking reagents were delivered to each of the discs in eachspine specimen by 2 to 3 injections into one lateral side of the spine.Each injection was comprised of 1 cc of reagent. A 26 gauge hypodermicneedle was used. The treated segments were allowed to sit in a closedcontainer wrapped in moist paper towels for 36 hours prior to finaltesting. After testing, the discs were cut transversely to visuallydocument the region of the tissue contacted by the reagents.

Resistance to lateral bending and lateral bending stability wereassessed by two measures, one elastic-plastic, the other viscoelastic.The first was the neutral zone (low-load) bending stiffness evidenced bythe amount of deformation from 0.1 to 100 N of deforming force. Thesecond was the hysteresis or bending energy lost or not stored by thetissues. Less hysteresis, or a lower percentage of hysteresis comparedto strain energy or the amount of bending energy stored and returned,corresponds to greater capacity to bounce back from a bend rather thanremain in the deformed position. It also reflects a more elastic,spring-like response as compared to a more viscous response.

The injections effectively distributed the crosslinking reagents toapproximately one-half of the disc annulus, right or left half. SeeTable 3. The neutral zone bending stiffness was consistently increasedby treatment only when the treated side was in tension. The averagemagnitude of stiffness increase was 12% with a 26% increase in the caseof 0.66% genipin plus 0.1% proanthocyanidin treatment. The hysteresiswas consistently decreased by treatment only when the treated side wasin tension. The average decrease in hysteresis was 31% with a 37%decrease in the case of 0.66% genipin plus 0.1% proanthocyanidintreatment. TABLE 3 Specimen # Side Max Max Loss of Change in Stiffness:Change in Stiffness: Side Up Treatment Treated Hysteresis DisplacementLoad Hysteresis Compression side Tensionon side 1L Control 87.21 6.87899.7 1L 0.50G L 97.47 8.381 99.0 −22% 1R Control 170.73 8.860 98.7 1R0.50G L 92.91 8.822 96.6 46% 0.43%   2L Control 64.41 3.463 99.3 2L0.50G L 47.80 3.873 97.6 −12% 2R Control 47.76 3.884 98.3 2R 0.50G L40.28 3.573 101.1 16%  8% 3L Control 80.70 7.116 100.4 3L 0.33G L 58.795.041 99.7 29% 3R Control 78.52 5.951 100.0 3R 0.33G L 50.67 4.924 97.635% 17% 4L Control 61.97 5.62 101.1 4L 0.66G 49.88 5.259 99.3 20%  6% 4RControl 63.65 5.359 98.7 4R 0.66G 50.92 4.931 99.3 8% 5L Control 41.583.511 100.7 0.66G + 0.1P 5L A 49.87 4.049 101.4 −15% 5R Control 74.894.683 100.4 0.66G + 0.1P 5R A 47.08 3.460 100.4 37% 26% Average 31% −2%12%

These results demonstrate that crosslink augmentation with minimallynon-toxic crosslinking reagents effectively reduces instability ofintervertebral discs toward deforming forces as is expected in scolioticspines. The stabilizing effect was observed to be greater with the 0.66%genipin plus 0.1% proanthocyanidin treatment. Consequently, by reducingthe viscoelastic dissipation of bending energy and increasing thebounce-back of the discs (lowered hysteresis) and by increasing thebending stiffness in the direction that puts the treated side of thespine in tension, injectable non-toxic crosslink augmentationeffectively resists scoliotic curve progression as well as otherprogressive spinal deformities.

EXAMPLE 4

By measuring the change in hydration of different regions of theintervertebral disc (nucleus pulposus, inner annulus, and outer annulusfibrosus) prior to and after periods of soaking, sustained compressiveloading, and resoaking, the fluid flux to and from different regions canbe determined. By comparing these measurements between control discs anddiscs treated with crosslinking reagents known to have minimalcytotoxicity, we see the effect of crosslinking treatment on fluid fluxand permeability.

A total of 24 calf (4 month old bovine) intervertebral discs were usedfor this study. Water content of three different areas of the discaltissue were tested—the nucleus pulposus, inner annulus fibrosus andouter annulus fibrosus. Hydration change was determined by weighing thespecimen using a micro-balance (sensitivity: 0.1 mg). Water content (M)was calculated as:M=(Wet Weight-Dry Weight)/Wet Weight=g H2O/g Wet Weight

The drying procedure consisted of putting the specimens in the oven witha controlled temperature of 90.degree C. for 24 hours.

The specimens were separated into four tests:

1. Group A: Three specimens were in this group. It served as a controlgroup. The specimens were soaked in PBS (phosphate buffered saline) for1 day and then the hydration analysis was performed.

2. Group B1: Four specimens were in this group. In addition to the oneday PBS soaking, the specimens soaked in PBS for 2 more days as acontrol and then the hydration analysis was performed.

Group B2: Five specimens were in this group. In addition to the one dayPBS soaking, the specimens were soaked in 0.33% genipin solution for 2days and then the hydration analysis was performed.

3. In group C, a small daytime amount of constant compressive loading(creep) was simulated.

C1: Three specimens were in this group. The specimens were soaked in PBSfor 3 days and then 750N of compression was applied by a materialstesting machine for 1 hour. The disc was compressed in a 5 degree offlexion posture produced by two rollers attached to the loading ram ofthe materials testing machine. The hydration analysis was performedimmediately after the creep loading.

C2: Three specimens were in this group. The specimens were soaked in0.33% Genipin solution for 2 days after 1 day of PBS soaking andperformed identical creep loading with 750N compressive load. Thehydration analysis was performed immediately after the creep loading.

4. In group D, the imbibition of water following a period of compressiveloading that typically occurs in the night time as a person is in arecumbent posture was simulated.

D1: The specimens were soaked in PBS solution for 3 days and then 1 hourof creep loading at 750 N was applied. After the creep loading, thespecimens were placed in a container in 1 PBS for one more day followedby the hydration analysis.

D2: Three specimens were included and were soaked in 0.33% genipinsolution for 2 days after one day of PBS soaking. A creep load of 750Nfor one hour was then applied. The specimens were put in PBS for anotherday followed by the hydration analysis.

See Table 4. In general, creep loading expels fluid out of the tissuesand after creep re-absorption of fluid occurs. The result pertinent tothe present invention was that there was a combined 64% increased fluidflow into and out of the central nucleus region in the genipincrosslinking reagent treated discs compared to controls. TABLE 4 ControlGenipin % increase Gr B1 Gr B2 Gr C1 Gr C2 Gr D1 Gr D2 Flux Flux byGenipin Inner 0.768771 0.762891 0.745779 0.739397 0.808709 0.0.8166690.08592 0.10077 17.3% AF Outer 0.723259 0.726776 0.696626 0.6924040.720096 0.710972 0.050010 0.05294 5.7% AF NP 0.834041 0.831405 0.8259980.816964 0.848403 0.852357 0.03045 0.04983 63.7%

These results demonstrate that augmentation of crosslinking ofintervertebral disc tissue resulted in an increased fluid flow into andout of the central region of the intervertebral disc. This increasedfluid flux to the disc nucleus indicates that this treatment effects anincrease of nutrients supplied to cells in the central region of thedisc as well as an increased removal of cell and matrix waste products.

EXAMPLE 5

Ten annulus fibrosus circumferentially aligned specimens from bovinelumbar (T12-L5) intervertebral discs were divided into two groups andeither soaked two days in a 0.15M PBS or a 0.15M PBS plus 0.33% genipinsolution. A custom loading fixture attached to a uniaxial materialstesting system was designed to hold an annulus specimen perpendicular tothe travel of a metal cylinder placed in a pre-existing radial cut. Thecylinder was then pulled radially (perpendicular to lamellae) towardsthe external layer. Time dependent force and displacement data wasacquired simultaneously. Tearing resistance was quantified in two ways:peak force normalized by specimen thickness and tearing energy per areaof tear. Differences between groups were analyzed using the Mann-Whitneynon-parametric test.

Normalized peak force was 23.9 and 42.7 N/mm for control and genipincrosslinks groups respectively (p=0.076). Total energy per area of tearwas 19.0 and 40.2 mJ/mmˆ2 for control and genipin groups (p=0.047, FIG.1).

Crosslink augmentation of the extracellular matrix of the annulusfibrosus was found to approximately double the peak force and the totalenergy required to propagate a radial tear. Injectable crosslinkaugmentation of the annulus fibrosus can prevent or slow down thedegradation leading to loss of the contents of the disc's centralregion, whether it involves herniation of nucleus pulposus or expulsionof nucleus replacement devices or materials.

EXAMPLE 6

One can perform a primary, conservative nonsurgical treatment of apatient suffering from back pain and possibly showing MRI orradiographic signs of disc degeneration by injecting an effective amountof crosslinking agent, such as 400 mM L-Threose in saline (0.15M) or asolution comprised of 200 mM methylglyoxal in saline or a solution of200 mM glyoxal or a solution 200 mM EDC or a solution comprised of50-100 μg lysyl oxidase in a 0.1 M urea saline solution or a solutioncomprised of 50 μg/ml human recombinant transglutaminase in saline, or asolution comprised of 200 μg/ml of purified animal livertransglutaminase in saline, to the affected disc. This treatment can beperformed in order to stabilize the degenerated intervertebral joint, toincrease the durability (fatigue resistance) and tear resistance of theaffected disc tissues, to improve nutritional flow to and waste productflow from the degraded disc, to enable rehydration of the disc and asubsequent increase in disc height, to change the load transmissionthrough the afflicted disc, to prevent further degenerative changes tothe disc, to potentially prevent migration and herniation of discmaterial with normal load bearing, to potentially relieve pressure on aneural element external to the disc, to potentially relieve pressure onnerve endings in the disc, and to reduce the incidence of back painepisodes. The crosslinking agent injection can be accompanied bystandard injections of pain medication or steroids if deemed appropriateby the physician.

EXAMPLE 7

One can treat a patient who has undergone installation of spinalinstrumentation to minimize consequent deformities or adjacent segmentdegeneration by treating the whole or part of the adjacentintervertebral disc with a crosslinking agent, such as 400 mM L-Threosein saline (0.15M) or a solution comprised of 200 mM methylglyoxal insaline or a solution of 200 mM glyoxal or a solution 200 mM EDC or asolution comprised of 50-100 μg lysyl oxidase in a 0.1 M urea salinesolution or a solution comprised of 50 μg/ml human recombinanttransglutaminase in saline, or a solution comprised of 200 μg/ml ofpurified animal liver transglutaminase in saline. Immediately afterinstallation of surgical apparatus or within a few days after surgerythe crosslinking agent can be injected into the whole disc adjacent tothe instrumented levels. Fluoroscopic or other imaging means can be usedto deliver the crosslinking agent to the selected tissues. In the caseof a long, multilevel thoracolumbar stabilization, the inferiorlyadjacent (caudal) lumbar disc can be treated to prevent loss of naturallordosis, and the superiorly adjacent (towards the head). In the case ofa single or multi-level fusion or non-fusion (such as artificial discimplantation or dynamic stabilization) the adjacent disc levels can betreated to prevent accelerated intervertebral joint degeneration(adjacent disc syndrome) at that level.

EXAMPLE 8

If a patient is suffering from grade I spondylolisthesis (25% or lessslip of one vertebra relative to an adjacent vertebra), neurologicalproblems including pain or weakness in the legs, or back pain, whereconventional surgical options may dictate performing a laminectomy todecompress the neural structures, one can treat the patient with thesame laminectomy procedure combined with injections of a crosslinkingreagent, such as 400 mM L-Threose in saline (0.15M) or a solutioncomprised of 200 mM methylglyoxal in saline or a solution of 200 mMglyoxal or a solution 200 mM EDC or a solution comprised of 50-100 μglysyl oxidase in a 0.1 M urea saline solution or a solution comprised of50 μg/ml human recombinant transglutaminase in saline, or a solutioncomprised of 200 μg/ml of purified animal liver transglutaminase insaline, into the partially “slipped” disc, either to the posterioraspect of the disc or to the entire annulus to minimize consequentprogression of the deformity. In such a case, prior to, at the sametime, or subsequent to the decompression procedure (laminectomy),according to the preference of the physician administering thetreatment, multiple injections of a preferred, non-toxic crosslinkingagent can be performed through a single or multiple injection sites. Thepatient should be instructed to avoid strenuous activities for a periodof a few days. The internal stabilization afforded by the crosslinkingtreatment can be augmented by other conservative measures such asexternal bracing according to the physician's judgment. If administeredprior to any decompression procedure, the crosslinking treatment canprovide stability to the spine in the region of the slipped disc thuspotentially avoiding the need for surgery including surgicaldecompression.

EXAMPLE 9

This is an example of in vivo animal testing of a hybrid treatmentincluding implantation of autologous stem cells and crosslinkingtreatment. Muscle cell derived autologous stem cells can be injectedinto the nucleus of mechanically degraded rat-tail intervertebral discsof four groups of Sprague-Dawley rats. One of the groups can have aneffective non-toxic crosslinking reagent such as 400 mM L-Threose insaline (0.15M) or a solution comprised of 200 mM methylglyoxal in salineor a solution of 200 mM glyoxal or a solution 200 mM EDC or a solutioncomprised of 50-100 μg lysyl oxidase in a 0.1 M urea saline solution ora solution comprised of 50 μg/ml human recombinant transglutaminase insaline, or a solution comprised of 200 μg/ml of purified animal livertransglutaminase in saline, injected into the target discs prior toimplantation of the stem cells. Another group can have the crosslinkingreagent injected into the target discs at approximately the same timethat the stem cells are implanted. A third group can have the targetdiscs treated with crosslinking 1 day following implantation of the stemcells. The fourth group can have the stem cells implanted with noaccompanying crosslinking treatment. Prior to treatment, each candidatedisc can receive 1 to 2 weeks of repetitive mechanical loading(compression bending to 1.2 MPa) to degrade the discs as documented byprior experiments. Following treatment the rats can be maintained for anadditional 2, 4, 8, or 16 weeks prior to sacrifice. Subsequent tosacrifice histochemical (quantify collagen I/II & proteoglycan matrixcontent, apoptotic cells, and inflammatory cells), mechanical, anabolic(Real-time PCR analysis will quantify collagen-1a1, collagen-2a1 andaggregan expression) and catabolic (MMP-3, MMP-13, ADAMTs-4 mRNAmarkers) assays can be performed on the disc tissue to document andquantify the efficacy, viability and duration of the cell-basedtreatment technique for the crosslinked and noncrosslinked groups overthe 4 time periods. Crosslink augmentation of the disc should improvestem cell viability and duration of effects, regeneration and repair ofdegraded extracellular tissues, disc viscoelastic and elastic-plasticmaterial properties, and joint stability.

EXAMPLE 10

One can treat a patient who has undergone, or will undergo, a cell orcytokine (growth factor) based treatment of a degenerated disc(including but not limited to implantation of stem cells of anyderivation, gene-therapy delivery of growth factors, implantation ofmatrices with attached growth factors, direct application of growthfactors, implantation of transplanted tissues or cells, implantation ofxenograft tissues or cells) with an adjunct treatment of the disc(preferably including endplates and annulus) by injecting a crosslinkingagent, such as 400 mM L-Threose in saline (0.15M) or a solutioncomprised of 200 mM methylglyoxal in saline or a solution of 200 mMglyoxal or a solution 200 mM EDC or a solution comprised of 50-100 μglysyl oxidase in a 0.1 M urea saline solution or a solution comprised of50 μg/ml human recombinant transglutaminase in saline, or a solutioncomprised of 200 μg/ml of purified animal liver transglutaminase insaline into or adjacent to the targeted areas (one would expectinjection of the superior and inferior aspects of the disc nucleus andinner annulus to promote contact between the crosslinking agents and thecartilaginous endplates of the disc). This treatment would beadministered in order to affect an increase in nutrient flow to thecells in the disc, to increase waste product removal from the cells, toincrease the regenerative and repair capabilities of the cells, and bythis to improve the efficacy of the cell or cytokine based treatment,and to improve the viability of cells in the disc, and by this tosustain the augmented biological activity and increase the duration ofbeneficial activities.

EXAMPLE 11

One can treat a patient who has undergone, or will undergo, a nucleusreplacement treatment of a moderately degenerated disc (including butnot limited to implantation of nucleus replacement material or device)with an adjunct treatment of the disc (preferably including endplatesand annulus) by injecting a crosslinking agent, such as 400 mM L-Threosein saline (0.15M) or a solution comprised of 200 mM methylglyoxal insaline or a solution of 200 mM glyoxal or a solution 200 mM EDC or asolution comprised of 50-100 μg lysyl oxidase in a 0.1 M urea salinesolution or a solution comprised of 50 μg/ml human recombinanttransglutaminase in saline, or a solution comprised of 200 μg/ml ofpurified animal liver transglutaminase in saline into all of theremaining disc annulus material and into or adjacent to thecartilaginous endplates of the disc (one would expect injection of thesuperior and inferior aspects of the disc nucleus region and innerannulus region to promote contact between the crosslinking agents andthe cartilaginous endplates of the disc). This treatment would beadministered in order to affect an increase in annulus tear resistanceand resist fissuring in the annulus and through the cartilaginousendplates, to prevent migration or extrusion or expulsion of implantedmaterials and devices and by that to improve the efficacy of the nucleusreplacement treatment, to maintain the long term integrity of theannulus and disc, to maintain augmented disc height, and by this toreduce compression of neural tissues and to reduce pain for longerduration post treatment.

EXAMPLE 12

This is an example of biomechanical testing of cadaveric intervertebraljoints after crosslinking treatment.

Individual calf and human cadaveric thoracolumbar intervertebral jointspecimens can be subjected to repetitive combinedflexion-compression-anterior shear loading in order to simulate akyphotic deformity of an intact spinal segment adjacent to a fused orotherwise instrumented segment. Four treatment groups can be comparedwith untreated controls. Each group contains 5 or more cadavericspecimens from similar regions of thoracolumbar spines. The firsttreatment group can be treated in the posterior half of the annulus byinjections with a solution comprised of 400 mM L-Threose in saline(0.15M). The total volume injected in each disc of this group can be 1ml. The second treatment group can be treated across the entire disc byinjections with a solution of 400 mM L-Threose in saline. The thirdtreatment group can be treated across the whole disc by injections witha solution comprised of 200 mM methylglyoxal in saline. The fourthtreatment group can be treated across the whole disc by injections witha solution comprised of 50-100 μg lysyl oxidase in saline. The totalvolume injected in these discs can be 3 ml. The preventative effects ofthe different crosslinking treatments can be compared by evaluatingamounts of unrecovered deformation subsequent to crosslink treatment andapplication of deforming loads. One thousand cycles of combined loadingcan be applied after which permanent deformation is measured using anoptoelectronic position measurement system. Peak loads and moments canbe uniform between groups. Peak moment can be between 1 Nm and 3 Nmdepending on the spinal level (i.e. upper thoracic being 1 Nm, lumbarbeing 3 Nm). Peak shear load can be 200 N and peak compressive load canbe 500 N in lumbar intervertebral levels. Peak shear can be 100 N andpeak compression can be 250 N in upper thoracic levels. The permanentdeformation measurements should demonstrate a decreased amount ofdeformation in the crosslinked specimens on average compared to theuntreated discs.

The invention has been described in terms of certain preferred andalternate embodiments which are representative of only some of thevarious ways in which the basic concepts of the invention may beimplemented. Certain modification or variations on the implementation ofthe inventive concepts which may occur to those of ordinary skill in theart are within the scope of the invention and equivalents, as defined bythe accompanying claims.

LIST OF REFERENCES

The following publications are hereby incorporated by reference:

List of References

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1. A method of treatment of native, non-denatured tissue to increaseresistance to tearing, fissuring, rupturing, and/or delamination,comprising the step of: contacting at least a portion of the tissue withan effective amount of a reagent that increases crosslinks in thetissue.
 2. The method of claim 1 in which the tissue is fibrouscollagenous tissue.
 3. The method of claim 2 in which the fibrouscollagenous tissue is the annulus fibrosus of an intervertebral disc orknee meniscus.
 4. The method of claim 1 in which there is more than onecrosslinking agent.
 5. The method of claim 1 in which the reagent isnon-toxic.
 6. The method of claim 1 in which the crosslinking reagentcontains one or more of the following agent concentrations: at least0.001% (0.01 mg/ml) of human recombinant transglutaminase; at least0.01% (0.1 mg/ml) of purified animal liver transglutaminase; at least0.25% genipin; at least 0.1% proanthrocyanidin; at least 100 mM EDC; atleast 50 mM non-enzymatic agent; or at least 0.001% lysyl oxidase. 7.The method of claim 6 in which the agent is a non-enzymatic agent. 8.The method of claim 7 including the reagent contains an oxidant, abuffer, or a combination thereof.
 9. The method of claim 8 in which theoxidant is hydrogen peroxide, sodium percarbonate, sodium borate, anamino acid hydroperoxide, or perborate, and wherein the buffer is sodiumbicarbonate or phosphate.
 10. The method of claim 6 in which thenon-enzymatic agent is one or more of the following agentconcentrations: at least 100 mM ribose; at least 100 mM L-threose; atleast 50 mM methylglyoxal; or at least 50 mM glyoxal.
 11. The method ofclaim 6 in which the agent is in a 0.1 M urea solution.
 12. The methodof claim 6 in which the crosslinking agent is in a substantiallynon-cytotoxic carrier medium.
 13. The method of claim 12 in which the pHof the carrier medium is adjusted to be substantially the same as thehealthy tissue environment.
 14. The method of claim 13 in which thecarrier medium is a phosphate buffered saline solution.
 15. The methodof claim 2 in which the fibrous collagenous tissue is the annulusfibrosus of an intervertebral disc and the reagent is effective toprevent displacement of the central portion of the disc posteriorly orposterolaterally.
 16. The method of claim 2 in which the fibrouscollagenous tissue is the annulus fibrosus of an intervertebral disc andthe step of: contacting the tissue with the reagent is at the same timeor prior to or after implantation of a nucleus augmentation orreplacement device.